The need for stable proteins and polypeptides for many applications is continuously increasing. This is particularly pronounced for therapeutic proteins in the pharmaceutical field. For the ease of both manufacturers and final users aqueous protein solutions are often the preferred form of administration. Moreover this is their common natural form that allows hydrated, three-dimensional folded complex formation. This conformation is generally reported as tertiary structure and its integrity is of vital importance for maintaining the biological activity of proteins. The irreversible loss of tertiary structure of proteins is referred as denaturation and causes inactivation. Because proteins and polypeptides in solution are exposed to many stresses which can cause physical (denaturation) and chemical (i.e. reactions such as hydrolysis, deamidation etc. . . . ) degradation, very often the development of liquid formulations is precluded. Presently, the most common way to achieve protein stability is the removal of water by suitable processes such as freeze-drying or spray-drying. However, both of these techniques (ref. “Formulation and Delivery of Proteins and Peptides” J. L. Cleland and R. Lan ger American Chemical Society, Washington, D.C. 1994) can induce protein unfolding. In particular with regard to lyophilisation protein unfolding can occur either during the initial freezing step or during acute dehydration by sublimation.
Concerning spray drying, thermal degradation, low efficiency, low yield and high levels of residual moisture are the main limitations of the technique.
Another problem is the difference in long term stability of analogous formulations obtained by different drying processes. In fact, depending on the dehydration method, the protein may assume different three-dimensional structures with the same initial biological activity but different shelf life.
The stabilising effect of carbohydrates and in particular of trehalose on proteins during freezing and dehydration is well documented (“Formulation and Delivery of Proteins and Peptides” J. L. Cleland and R. Lan ger American Chemical Society, Washington, D.C. 1994 and “Freeze-Drying/Lyophilization of Pharmaceutical and Biological Products” L. Rey and J. C. May, Marcel Dekker, Inc. New York 1999). Although many sugars can prevent protein damage during dehydration, the products often have a short shelf life at room temperature due to the Maillard reaction. Stability at room temperature can be improved using non reducing sugars such as sucrose and trehalose.
British Patent Application GB 2009198 discloses lyophilisation of meningococcal polysaccharide and trehalose; GB 2126588 discloses the stabilisation of tumour necrosis factor (TNF) to lyophilisation and freezing by including either a non-ionic surfactant or trehalose (or another sugar); and Japanese Patent Application J 58074696 discloses the freeze-drying of ATP in presence of trehalose.
Preparations of alkaline phosphatase containing trehalose are reported to maintain their activity after freeze-drying and to maintain about 70% of the initial activity after 84 days/storage at 45° C. (A. W. Ford et Al., J. Pharm. Pharmacol. 1993, 45: 86-93). Although lyophilisation is still the main process used for drying proteins, several precautions must be taken in order to avoid the damage that severe stressing phases such as freeze-thawing and drying can cause. In fact, during the first step in freeze-dried protein formulation, a correct choice of conditions (pH, ionic strength, presence of stabilisers, etc. . . . ) guarantees the best protection against protein unfolding and inactivation. Many excipients such as sugars, aminoacids, polymers, surfactants specific ligands (substrates, co-factors, allosteric modifiers etc. . . . ) are known to stabilise proteins during freeze-drying and have been named “lyoprotectants”. Among them, carbohydrates and in particular disaccharides such as sucrose and trehalose have been widely studied. The stabilising mechanism of these compounds as well as other stabilisers has not been completely clarified. However, an effective lyoprotectant must maintain stability during both freeze-thawing and drying. Since the protein environment is aqueous during much of the freezing process, solutes that stabilise the native conformation in aqueous solutions are very often effective as protein cryoprotectants. Carbohydrates and some aminoacids are examples. Arakawa et Al. (J. Pharm. Res. 1991, 8, 285-291) reported that such solutes tend to be excluded from the surface of protein while in aqueous solution. The thermodynamic consequence of such phenomenon is the stabilisation of protein native conformation.
Stability during drying and storage is best explained by both the water substitution and the vitrification hypotheses. The first states that stabilisers interact with the protein as water does by replacing the removed water and accounts for the thermodynamic control of drying process. The latter states that stabilisers are good glass formers and remain amorphous during and after drying so that they mechanically immobilise proteins inside a glassy matrix. This is a purely kinetic argument that applies equally well to both drying and storage stability. (“Freeze-Drying/Lyophilization of Pharmaceutical and Biological Products” L. Rey and J. C. May, Marcel Dekker, Inc. New York 1999).
Hence, referring to the above stabilisation hypothesis of dried proteins, it can be postulated that vitrification is one of the main issues for long term stability. The use of spray-drying for protein desiccation has been less investigated. Although fine amorphous particles can be produced, this process requires warm air as a drying force that can lead to protein thermal degradation. Moreover, low efficiency, low yield and high levels of residual moisture are other limitations.
Another reported technique for drying proteins which should avoid inactivation is air dehydration at room temperature. U.S. Pat. No. 4,891,319 of Quadrant Bioresources Ltd (UK) discloses the preservation of several proteins and other macromolecules at 37-40° C. by drying in presence of trehalose at atmospheric pressure.
The use of supercritical fluid technology has also been reported as a useful method for obtaining proteins as dry fine micro-particles. The main advantages of this technique are the possibility of maintaining the protein in a favourable aqueous environment before a rapid precipitation in order to minimise denaturation and the process length which is shorter than freeze-drying and less expensive.
S. P. Sellers et Al. (J. Pharm. Sci., 2001, 90, 785-797) report a dehydration method for protein powder production based on supercritical CO2-assisted nebulization. This technique can be assimilated to spray-drying; in fact supercritical CO2 is used for enhancing solution nebulization and not as an anti-solvent solvent for solute precipitation. The GAS (Gas Anti-Solvent recrystallization) process to form protein microparticles is reported by Debenedetti (U.S. Pat. No. 6,063,910). In this case the protein solution is sprayed through a laser drilled platinum disc with a diameter of 20 μm and a length of 240 μm inside the particle formation vessel previously filled by supercritical fluid which is introduced by a different inlet. This technique has been used to form particles of catalase and insulin (0.01% w/v) from ethanol/water (9:1 v/v) solutions using carbon dioxide as the supercritical fluid. In this process, the supercritical fluid inlet is not optimized: the solution injection occurs in an almost static atmosphere of supercritical fluid, with low turbulence. Hanna M. and York P. (WO96/00610) proposed a new method and a new apparatus to obtain very small particles by a specific supercritical fluid technique named SEDS (Solution Enhanced Dispersion by Supercritical Solution).
The process is based on a new coaxial nozzle: the solution expands through a capillary inlet, supercritical fluid expands through an external coaxial pathway with a conical shaped end. The mixing between supercritical fluid and solution occurs in the conical zone. They also propose the use of a three way nozzle: a modifier can be fed in order to improve the mixing.
They applied the SEDS technology to precipitation of small particles of water soluble compounds, such as sugars (Lactose, Maltose, Trehalose and Sucrose) and proteins (R-TEM beta-lactamase). Co-precipitation of proteins and stabilisers is not mentioned nor exemplified therein.
Moreover, the same inventors (WO01/03821) describe an improved precipitation method using the same apparatus but feeding to the particle formation vessel a supercritical fluid and two immiscible solvents. This method allows co-precipitation of two or more solutes dissolved in the two immiscible solvents. The fluids inlet is formed by a coaxial nozzle wherein contact between the two solvents occurs shortly before their dispersion by the supercritical anti-solvent, avoiding the precipitation solutes inside the nozzle. However this method permits the formation of homogeneous co-precipitates; it is generally useful when two solutes with different polarity must be processed. Moreover, if this is used for an aqueous solution, the second solvent must be at least partly soluble in water so that it allows the water to disperse in the supercritical anti-solvent. This step is necessary to permit water-soluble solute precipitation. Co-precipitation of proteins and stabilisers is not described in this document.
Walker (WO01/15664) discloses a method for co-formulating an active (preferably a pharmaceutically active) substance and an oligomeric or polymeric excipient in which an amount between 80 and 100% of the active substance is in amorphous as opposed to crystalline form. In these formulations the active substances are more stable compared to the crystalline forms when stored at temperature between 0 and 10° C. Only the co-formulation of a pharmaceutical active substance with an oligomeric or polymeric excipient is disclosed and there is no mention of protein stabilisation in this document. Protein stabilisation is therefore achieved in the art through freeze-drying and spray-drying. The co-precipitation of proteins with stabilisers using supercritical fluids has not been described before and it is the object of the present invention.
We have now found a method of producing stable dry protein microparticles by co-precipitation with a stabiliser using supercritical fluids. Preferred stabilisers are carbohydrates, aminoacids, surfactants and polymers. More preferably the stabilizer is a sugar, most preferably trehalose.
Co-precipitation allows intimate interactions between the protein/stabiliser molecules and an optimal weight/weight ratio exists for each couple protein/stabiliser.
In fact since there is no freeze-thawing there is no need for cryoprotection. Moreover, although the nature of protein/stabiliser interactions has to be better clarified, in the present case the stabiliser plays the essential role of improving storage stability rather than that of retaining protein activity during drying. In fact, precipitation by a supercritical fluid allows by itself protein particle production without denaturation during the drying process.